PROJECT SUMMARY Leukapheresis is a specialized medical procedure during which patient's whole blood (WB) is passed through the extracorporeal circuit of an apheresis machine, which extracts white blood cells (WBCs) from WB, and returns red blood cells (RBCs) and platelets (PLTs) back to the patient. The separation of WBCs from patient's blood via leukapheresis is the key initial step for an increasing number of highly effective cell-based treatments for some of the most devastating hematologic and immune system disorders affecting millions of adults and children worldwide. Currently, leukapheresis is performed using centrifugation-based apheresis machines, which have a substantial extracorporeal volume (ECV). Although well-tolerated by most adults and older children, leukapheresis in young children weighing less than about 10 kg (or 22 lbs) is technically challenging and clinically risky. Because ECV represents a particularly large fraction of their total blood volume (TBV), these vulnerable patients experience a significantly higher incidence of hypotension, symptomatic hypocalcemia, allergic reactions, catheter-related thrombosis, infections, severe anemia and even death. There is currently no practical alternative to adult-size apheresis machines for performing leukapheresis in neonates and low-weight infants. To address this significant limitation, we will develop and validate novel high-throughput microfluidic devices with very low void volume, capable of separating WBCs from WB with volumetric throughput and efficiency sufficiently high to ultimately enable centrifugation-free, low-ECV leukapheresis. These devices will utilize new cell separation technology (`controlled incremental filtration', or CIF) which we have previously applied to separating WBCs from concentrated blood cell suspensions with high efficiency, minimal RBC and PLT loss, and at flow rates on par with conventional leukapheresis. Here we will apply this approach to separating WBCs directly from WB by completing three complementary aims with scope ranging from iterative design optimization and validation work, to testing the performance of the CIF-based leukapheresis devices in an animal model. First, we will optimize the CIF design parameters to maximize WBC separation, while minimizing RBC/PLT losses and device fluidic resistance when processing WB. Second, we will multiplex individual CIF device modules into full- scale device prototypes, optimize their operation in the recirculation regime, and validate their ability to process large volumes of WB in vitro. Third, we will comprehensively evaluate device performance and the effect of CIF- based processing on blood cell properties in a mouse model. Detailed blood cell counts and markers of cell activation and damage will be measured throughout the project to aid the iterative design process and to validate the use of CIF technology for leukapheresis. By completing this research, we will develop functional device prototypes and generate pivotal data to support further testing in a pre-clinical model (porcine), before finalizing the device design for manufacturing from thermoplastic and clinical testing in human subjects.